Abstract

The FRAP reagent contains 2,4,6-tris(2-pyridyl)-s-triazine, which forms a blue–violet complex ion in the
presence of ferrous ions. Although the FRAP (ferric reducing/antioxidant power) assay is popular and has
been in use for many years, the correct molar extinction coefficient of this complex ion under FRAP assay
conditions has never been published, casting doubt on the validity of previous calibrations. A previously
reported value of 19,800 is an underestimate. We determined that the molar extinction coefficient was
21,140. The value of the molar extinction coefficient was also shown to depend on the type of assay
and was found to be 22,230 under iron assay conditions, in good agreement with published data. Redox
titration indicated that the ferrous sulfate heptahydrate calibrator recommended by Benzie and Strain,
the FRAP assay inventors, is prone to efflorescence and, therefore, is unreliable. Ferrous ammonium sulfate
hexahydrate in dilute sulfuric acid was a more stable alternative. Few authors publish their calibration
data, and this makes comparative analyses impossible. A critical examination of the limited number
of examples of calibration data in the published literature reveals only that Benzie and Strain obtained a
satisfactory calibration using their method